IL-1ß + TGF-ß2 dual-licensed mesenchymal stem cells have reduced major histocompatibility class I expression and positively modulate tenocyte migration, metabolism, and gene expression.

OBJECTIVE: The study objectives were to 1) determine the mesenchymal stem cell (MSC) surface expression of major histocompatibility complex (MHC) class I and transcriptome-wide gene expression changes following IL-1ß + TGF-ß2 dual licensing and 2) evaluate if IL-1ß + TGF-ß2 dual-licensed MSCs had a greater ability to positively modulate tenocyte function compared to naive MSCs. SAMPLE: Equine bone marrow-derived MSCs from 6 donors and equine superficial digital flexor tenocytes from 3 donors. METHODS: Experiments were performed in vitro. Flow cytometry and bulk RNA sequencing were utilized to determine naive and dual-licensed MSC phenotype and transcriptome-wide changes in gene expression. Conditioned media were generated from MSCs and utilized in tenocyte cell culture assays as a method to determine the effect of MSC paracrine factors on tenocyte function. RESULTS: Dual-licensed MSCs have a reduced expression of MHC class I and exhibit enrichment in functional pathways associated with the extracellular matrix, cell signaling, and tissue development. Additionally, dual-licensed MSC-conditioned media significantly improved in vitro tenocyte migration and metabolism to a greater degree than naive MSC-conditioned media. In tenocytes exposed to IL-1ß, dual-licensed conditioned media also positively modulated tenocyte gene expression. CLINICAL RELEVANCE: Our data indicate that conditioned media containing paracrine factors secreted from dual-licensed MSCs significantly modulates in vitro tenocyte function, which may confer benefits in vivo to healing tendons following injury. Additionally, due to reduced MHC class I expression in dual-licensed MSCs, this technique may also provide an avenue to provide an effective "off-the-shelf" allogenic source of MSCs.

A myristoylated alanine-rich C-kinase substrate (MARCKS) inhibitor peptide attenuates neutrophil outside-in ß2-integrin activation and signaling.

MARCKS is an actin and PIP2-binding protein that plays an essential role in neutrophil migration and adhesion; however, the molecular details regarding MARCKS function in these processes remains unclear. Neutrophil adhesion and migration also require the cell surface receptors ß2-integrins. We hypothesized that MARCKS inhibition would alter neutrophil ß2-integrin activation and signaling. We utilized a MARCKS-targeting peptide to inhibit MARCKS in inside-out and outside-in ß2-integrin activation in neutrophils. MANS-mediated MARCKS inhibition had no significant effect on inside-out ß2-integrin activation. MANS treatment significantly attenuated ICAM-1/Mn2+-stimulated static adhesion, cell spreading and ß2-integrin clustering, suggesting a role for MARCKS function in outside-in ß2-integrin activation. Additional work is needed to better understand the molecular mechanisms of MARCKS role in outside-in ß2-integrin activation and signaling.

Effect of gentamicin on CD3+ T-lymphocyte proliferation for treatment of equine recurrent uveitis: An in vitro study.

OBJECTIVE: The objective of the study was to determine the effect of gentamicin on CD3+ T-lymphocyte proliferation and cell viability using an in vitro cell culture model as a means of investigating the mechanism of action of low-dose intravitreal gentamicin injection. ANIMALS STUDIED: Three adult horses with no evidence of ophthalmic or systemic disease. PROCEDURE: Peripheral blood lymphocytes were treated with gentamicin at concentrations 37.5 µg/mL, 112.5 µg/mL, 187 µg/mL, 375 µg/mL, or 750 µg/mL then stimulated to proliferate with concanavalin A (ConA). 4',6-diamidino-2-phenylindole (DAPI) and carboxyfluoroscein succinimidyl ester (CSFE) were used as markers of cell viability and cell proliferation, respectively. Following 5-day culture, live cell counts and CSFE fluorescent intensity data were collected via automated cell count and flow cytometry. The experimental design was duplicated using preservative-free gentamicin and a proprietary brand formulation. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test. RESULTS: No statistically significant comparisons in CD3+ T-lymphocyte live cell counts and geometric mean fluorescent intensity of CSFE were identified between gentamicin concentrations or formulations. CONCLUSIONS: Gentamicin had no effect on equine peripheral blood CD3+ T-lymphocyte cell viability and proliferation in concentrations ranging from "safe" to "retinotoxic" in relation to intravitreal injection volumes. Low-dose intravitreal gentamicin may not suppress the Th1- and Th17-mediated immune response.

TGF-ß2 enhances expression of equine bone marrow-derived mesenchymal stem cell paracrine factors with known associations to tendon healing.

BACKGROUND: Mesenchymal stem cells (MSCs) secrete paracrine factors and extracellular matrix proteins that contribute to their ability to support tissue healing and regeneration. Both the transcriptome and the secretome of MSCs can be altered by treating the cells with cytokines, but neither have been thoroughly investigated following treatment with the specific cytokine transforming growth factor (TGF)-ß2. METHODS: RNA-sequencing and western blotting were used to compare gene and protein expression between untreated and TGF-ß2-treated equine bone marrow-derived MSCs (BM-MSCs). A co-culture system was utilized to compare equine tenocyte migration during co-culture with untreated and TGF-ß2-treated BM-MSCs. RESULTS: TGF-ß2 treatment significantly upregulated gene expression of collagens, extracellular matrix molecules, and growth factors. Protein expression of collagen type I and tenascin-C was also confirmed to be upregulated in TGF-ß2-treated BM-MSCs compared to untreated BM-MSCs. Both untreated and TGF-ß2-treated BM-MSCs increased tenocyte migration in vitro. CONCLUSIONS: Treating equine BM-MSCs with TGF-ß2 significantly increases production of paracrine factors and extracellular matrix molecules important for tendon healing and promotes the migration of tenocytes in vitro.

Interleukin-1ß in tendon injury enhances reparative gene and protein expression in mesenchymal stem cells.

Tendon injury in the horse carries a high morbidity and monetary burden. Despite appropriate therapy, reinjury is estimated to occur in 50-65% of cases. Although intralesional mesenchymal stem cell (MSC) therapy has improved tissue architecture and reinjury rates, the mechanisms by which they promote repair are still being investigated. Additionally, reevaluating our application of MSCs in tendon injury is necessary given recent evidence that suggests MSCs exposed to inflammation (deemed MSC licensing) have an enhanced reparative effect. However, applying MSC therapy in this context is limited by the inadequate quantification of the temporal cytokine profile in tendon injury, which hinders our ability to administer MSCs into an environment that could potentiate their effect. Therefore, the objectives of this study were to define the temporal cytokine microenvironment in a surgically induced model of equine tendon injury using ultrafiltration probes and subsequently evaluate changes in MSC gene and protein expression following in vitro inflammatory licensing with cytokines of similar concentration as identified in vivo. In our in vivo surgically induced tendon injury model, IL-1ß and IL-6 were the predominant pro-inflammatory cytokines present in tendon ultrafiltrate where a discrete peak in cytokine concentration occurred within 48 h following injury. Thereafter, MSCs were licensed in vitro with IL-1ß and IL-6 at a concentration identified from the in vivo study; however, only IL-1ß induced upregulation of multiple genes beneficial to tendon healing as identified by RNA-sequencing. Specifically, vascular development, ECM synthesis and remodeling, chemokine and growth factor function alteration, and immunomodulation and tissue reparative genes were significantly upregulated. A significant increase in the protein expression of IL-6, VEGF, and PGE2 was confirmed in IL-1ß-licensed MSCs compared to naïve MSCs. This study improves our knowledge of the temporal tendon cytokine microenvironment following injury, which could be beneficial for the development and determining optimal timing of administration of regenerative therapies. Furthermore, these data support the need to further study the benefit of MSCs administered within the inflamed tendon microenvironment or exogenously licensed with IL-1ß in vitro prior to treatment as licensed MSCs could enhance their therapeutic benefit in the healing tendon.

Defining the Profile: Characterizing Cytokines in Tendon Injury to Improve Clinical Therapy.

Cytokine manipulation has been widely used to bolster innate healing mechanisms in an array of modern therapeutics. While other anatomical locations have a more definitive analysis of cytokine data, the tendon presents unique challenges to detection that make a complete portrayal of cytokine involvement during injury unattainable thus far. Without this knowledge, the advancement of tendon healing modalities is limited. In this review, we discuss what is known of the cytokine profile within the injured tendinous environment and the unique obstacles facing cytokine detection in the tendon while proposing possible solutions to these challenges. IL-1ß, TNF-α, and IL-6 in particular have been identified as key cytokines for initiating tendon healing, but their function and temporal expression are still not well understood. Methods used for cytokine evaluation in the tendon including cell culture, tissue biopsy, and microdialysis have their strengths and limitations, but new methods and approaches are needed to further this research. We conclude that future study design for cytokine detection in the injured tendon should meet set criteria to achieve definitive characterization of cytokine expression to guide future therapeutics.

Adeno-Associated Virus-Mediated Overexpression of Interleukin-10 Affects the Immunomodulatory Properties of Equine Bone Marrow-Derived Mesenchymal Stem Cells.

Joint injury can cause posttraumatic inflammation, which if severe enough can lead to posttraumatic osteoarthritis (PTOA), a progressive and debilitating condition. Posttraumatic inflammation is characterized by an influx of T lymphocytes and upregulation of inflammatory cytokines and degradative enzymes by activated chondrocytes and synoviocytes. Intra-articular bone marrow-derived mesenchymal stem cell (BM-MSC) injection for the treatment of osteoarthritis (OA) has been of interest due to the immunomodulatory properties of these cells. Interleukin (IL)-10, a potent immunomodulatory cytokine, has also been investigated as an OA therapeutic. Therefore, the objective of this study was to evaluate the combinatorial effects of BM-MSCs and IL-10 in OA using a gene therapy approach. We hypothesized that BM-MSCs overexpressing IL-10 would have superior immunomodulatory effects leading to increased suppression of T cell proliferation and decreased production of proinflammatory cytokines, providing protection of the extracellular matrix (ECM) in a stimulated, co-culture OA model. Treatment groups included the following: untransduced BM-MSC, adeno-associated virus (AAV)-IL10-transduced BM-MSC, and AAV-null transduced BM-MSC, which were unstimulated or stimulated with IL-1ß/tumor necrosis factor-α (TNF-α). T cell proliferation was significantly decreased by the presence of BM-MSCs, especially when these BM-MSCs were AAV transduced. There was no significant difference in T cell suppression when cells were cultured with AAV-IL10-transduced or AAV-null transduced BM-MSCs. AAV transduction itself was associated with decreased synthesis of IL-1ß, IL-6, and TNF-α. Expression of IL-1ß and MMP13 was downregulated in AAV-transduced BM-MSCs and MMP13 expression was downregulated in cartilage explants co-cultured with AAV-transduced BM-MSCs. Despite mitigation of some proinflammatory cascades, rescue of ECM loss, as determined by glycosaminoglycan quantification and histological evaluation, did not occur in either AAV-IL10-transduced or AAV-null transduced co-cultures. Although IL-10 overexpression may enhance BM-MSC-mediated T cell suppression, we did not observe significant modulation of inflammation-driven cartilage degradation in cultures containing AAV-IL10-transduced BM-MSCs. AAV transduction itself does appear to affect paracrine signaling by BM-MSCs, which warrants further investigation.

TGF-ß2 Reduces the Cell-Mediated Immunogenicity of Equine MHC-Mismatched Bone Marrow-Derived Mesenchymal Stem Cells Without Altering Immunomodulatory Properties.

Allogeneic mesenchymal stem cells (MSCs) are a promising cell therapy for treating numerous diseases, but major histocompatibility complex (MHC)-mismatched MSCs can be rejected by the recipient's immune system. Pre-treating MSCs with transforming growth factor-ß2 (TGF-ß2) to downregulate surface expression of MHC molecules may enhance the ability of allogeneic MSCs to evade immune responses. We used lymphocyte proliferation assays and ELISAs to analyze the immunomodulatory potential of TGF-ß2-treated equine bone marrow-derived MSCs. T cell activation and cytotoxicity assays were then used to measure the in vitro cell-mediated immunogenicity. Similar to untreated MSCs, TGF-ß2-treated MSCs inhibited T cell proliferation and did not stimulate MHC-mismatched T cells to proliferate. Additionally, similar quantities of prostaglandin E2 and TGF-ß1 were detected in assays with untreated and TGF-ß2-treated MSCs supporting that TGF-ß2-treated MSCs retain their strong immunomodulatory properties in vitro. Compared to untreated MSCs, TGF-ß2-treated MSCs induced less T cell activation and had reduced cell-mediated cytotoxicity in vitro. These results indicate that treating MSCs with TGF-ß2 is a promising strategy to reduce the cell-mediated immunogenicity of MHC-mismatched MSCs and facilitate allogeneic MSC therapy.

Effects of continuous passage on the immunomodulatory properties of equine bone marrow-derived mesenchymal stem cells in vitro.

BACKGROUND: The immunomodulatory properties of mesenchymal stem cells (MSCs) have been studied extensively due to their increasing clinical application for tissue regeneration and repair following culture expansion. We have studied the effect of continuous passage on the immunomodulatory capacity of equine bone marrow-derived MSCs (BM-MSCs). Equine BM-MSCs were isolated and culture expanded to passage three, six, and nine (P3, P6, P9). Immunomodulatory properties of each passage were assessed using a T cell proliferation assay and cytokine synthesis following stimulation with interferon gamma (IFN-γ). RESULTS: Equine BM-MSCs maintained their primary cell morphology and immunophenotype throughout all passages. T cell proliferation was suppressed by all passages of BM-MSCs, compared to peripheral blood mononuclear cells (PBMCs) alone. There was no significant difference in suppression of T cell proliferation between P3, P6, and P9 BM-MSCs. All passages of BM-MSCs significantly increased cytokine synthesis in response to stimulation with IFN-γ. There were no significant differences in production of interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1) or regulate on activation, normal T cell expressed and secreted (RANTES) following stimulation with IFN-γ between P3, P6, and P9 BM-MSCs. P9 BM-MSCs had significantly increased production of tumor necrosis factor alpha (TNF-α), (IL-1ß), and (IL-10) compared to P3 BM-MSCs. Additionally, there was a significant increase in production of (IL-8) in P6 and P9 BM-MSCs in comparison to P3 BM-MSCs. CONCLUSIONS: Our findings demonstrate that culture expansion affects some of the immunomodulatory properties of BM-MSCs in vitro, which may suggest that MSCs isolated from a single collection of bone marrow may be culture expanded, but only those from lower passage numbers would be ideal for clinical application.

Cross-matching of allogeneic mesenchymal stromal cells eliminates recipient immune targeting.

Allogeneic mesenchymal stromal cells (MSCs) have been used clinically for decades, without cross-matching, on the assumption that they are immune-privileged. In the equine model, we demonstrate innate and adaptive immune responses after repeated intra-articular injection with major histocompatibility complex (MHC) mismatched allogeneic MSCs, but not MHC matched allogeneic or autologous MSCs. We document increased peri-articular edema and synovial effusion, increased synovial cytokine and chemokine concentrations, and development of donor-specific antibodies in mismatched recipients compared with recipients receiving matched allogeneic or autologous MSCs. Importantly, in matched allogeneic and autologous recipients, but not mismatched allogeneic recipients, there was increased stromal derived factor-1 along with increased MSC concentrations in synovial fluid. Until immune recognition of MSCs can be avoided, repeated clinical use of MSCs should be limited to autologous or cross-matched allogeneic MSCs. When non-cross-matched allogeneic MSCs are used in single MSC dose applications, presensitization against donor MHC should be assessed.

Pooled Platelet-Rich Plasma Lysate Therapy Increases Synoviocyte Proliferation and Hyaluronic Acid Production While Protecting Chondrocytes From Synoviocyte-Derived Inflammatory Mediators.

Platelet-rich plasma (PRP) preparations are being used with moderate success to treat osteoarthritis (OA) in humans and in veterinary species. Such preparations are hindered, however, by being autologous in nature and subject to tremendous patient and processing variability. For this reason, there has been increasing interest in the use of platelet lysate preparations instead of traditional PRP. Platelet lysate preparations are acellular, thereby reducing concerns over immunogenicity, and contain high concentrations of growth factors and cytokines. In addition, platelet lysate preparations can be stored frozen for readily available use. The purpose of this study was to evaluate the effects of a pooled allogeneic platelet-rich plasma lysate (PRP-L) preparation on equine synoviocytes and chondrocytes challenged with inflammatory mediators in-vitro to mimic the OA joint environment. Our hypothesis was that PRP-L treatment of inflamed synoviocytes would protect chondrocytes challenged with synoviocyte conditioned media by reducing synoviocyte pro-inflammatory cytokine production while increasing synoviocyte anti-inflammatory cytokine production. Synoviocytes were stimulated with either interleukin-1ß (IL-1ß) or lipopolysaccharide (LPS) for 24 h followed by no treatment or treatment with platelet-poor plasma lysate (PPP-L) or PRP-L for 48 h. Synoviocyte growth was evaluated at the end of the treatment period and synoviocyte conditioned media was assessed for concentrations of hyaluronic acid (HA), IL-1ß, tumor necrosis factor alpha (TNF-α), and interleukin-6 (IL-6). Chondrocytes were then challenged for 48 h with synoviocyte conditioned media from each stimulation and treatment group and examined for gene expression of collagen types I (COL1A1), II (COL2A1), and III (COL3A1), aggrecan (ACAN), lubricin (PRG4), and matrix metallopeptidase 3 (MMP-3) and 13 (MMP-13). Treatment of inflamed synoviocytes with PRP-L resulted in increased synoviocyte growth and increased synoviocyte HA and IL-6 production. Challenge of chondrocytes with conditioned media from PRP-L treated synoviocytes resulted in increased collagen type II and aggrecan gene expression as well as decreased MMP-13 gene expression. The results of this study support continued investigation into the use of pooled PRP-L for the treatment of osteoarthritis and warrant further in-vitro studies to discern the mechanisms of action of PRP-L.

Immunoprivileged no more: measuring the immunogenicity of allogeneic adult mesenchymal stem cells.

BACKGROUND: Autologous and allogeneic adult mesenchymal stem/stromal cells (MSCs) are increasingly being investigated for treating a wide range of clinical diseases. Allogeneic MSCs are especially attractive due to their potential to provide immediate care at the time of tissue injury or disease diagnosis. The prevailing dogma has been that allogeneic MSCs are immune privileged, but there have been very few studies that control for matched or mismatched major histocompatibility complex (MHC) molecule expression and that examine immunogenicity in vivo. Studies that control for MHC expression have reported both cell-mediated and humoral immune responses to MHC-mismatched MSCs. The clinical implications of immune responses to MHC-mismatched MSCs are still unknown. Pre-clinical and clinical studies that document the MHC haplotype of donors and recipients and measure immune responses following MSC treatment are necessary to answer this critical question. CONCLUSIONS: This review details what is currently known about the immunogenicity of allogeneic MSCs and suggests contemporary assays that could be utilized in future studies to appropriately identify and measure immune responses to MHC-mismatched MSCs.

Transforming Growth Factor-ß2 Downregulates Major Histocompatibility Complex (MHC) I and MHC II Surface Expression on Equine Bone Marrow-Derived Mesenchymal Stem Cells Without Altering Other Phenotypic Cell Surface Markers.

Allogeneic mesenchymal stem cells (MSCs) are a promising cell source for treating musculoskeletal injuries in horses. Effective and safe allogeneic therapy may be hindered, however, by recipient immune recognition and rejection of major histocompatibility complex (MHC)-mismatched MSCs. Development of strategies to prevent immune rejection of MHC-mismatched MSCs in vivo is necessary to enhance cell survival and potentially increase the efficacy and safety of allogeneic MSC therapy. The purposes of this study were to evaluate if transforming growth factor-ß2 (TGF-ß2) downregulated MHC expression on equine MSCs and to determine if TGF-ß2 treatment altered the phenotype of MSCs. Equine bone marrow-derived MSCs from 12 horses were treated with 1, 5, or 10 ng/ml TGF-ß2 from initial isolation until MHC expression analysis. TGF-ß2-treated MSCs had reduced MHC I and MHC II surface expression compared to untreated controls. TGF-ß2 treatment also partially blocked IFN-γ-induced upregulation of MHC I and MHC II. Constitutive and IFN-γ-induced MHC I and MHC II expression on equine MSCs was dynamic and highly variable, and the effect of TGF-ß2 was significantly dependent on the donor animal and baseline MHC expression. TGF-ß2 treatment did not appear to change morphology, surface marker expression, MSC viability, or secretion of TGF-ß1, but did significantly increase the number of cells obtained from culture. These results indicate that TGF-ß2 treatment has promise for regulating MHC expression on MSCs to facilitate allogeneic therapy, but further work is needed to maintain MHC stability when exposed to an inflammatory stimulus.

Effect of bone marrow-derived mesenchymal stem cells and stem cell supernatant on equine corneal wound healing in vitro.

BACKGROUND: We aimed to determine and compare the in vitro effects of autologous bone marrow-derived mesenchymal stem cells (BM-MSCs) and mesenchymal stem cell supernatant (MSC-Sp) on the wound healing capacity of equine corneal fibroblasts using a scratch assay. METHODS: Bone marrow aspirates and eyes were collected from normal, euthanized horses with subsequent isolation and culture of BM-MSCs and corneal stromal cells. Corneal stromal cells were culture-expanded in the culture well of transwell plates and then treated with an autologous BM-MSC suspension (dose: 2.5 × 105/100 µL media with the BM-MSCs contained within the insert well), MSC-Sp solution, or naive culture media (control) for 72 h. A linear defect in confluent cell cultures was created (i.e., corneal scratch assay) to assess the cellular closure ("healing") over time. Three representative areas of the scratch in each culture were photographed at each time point and the scratch area was quantitated using image analysis software (ImageJ). Media from the scratches were analyzed for various growth factors using human enzyme-linked immunosorbent assay (ELISA) kits that crossreact with the horse. RESULTS: There was a significant percentage decrease in the scratch area remaining in the BM-MSC and MSC-Sp groups compared to the control group. There was also a significant percentage decrease in the scratch area remaining in the BM-MSC group compared to the MSC-Sp group at 36 h post-scratch and all time points thereafter. The concentration of transforming growth factor (TGF)-ß1 in the media was significantly higher in the BM-MSC group compared to the control group. CONCLUSIONS: The significant decrease in scratch area in equine corneal fibroblast cultures treated with autologous BM-MSCs compared to MSC-Sp or control treatments suggests that BM-MSCs may substantially improve corneal wound healing in horses. MSC-Sp may also improve corneal wound healing given the significant decrease in scratch area compared to control treatments, and would be an immediately available and cost-effective treatment option.